The present invention relates to immunoreactive peptides that are homologous with the region encompassing amino acid positions 450 to 655 of glycoprotein II of varicella zoster virus. More particularly, the invention relates to peptides that correspond to amino acids 505 to 647, 517 to 597, 535 to 584 or 545 to 582 of the protein. The immunoreactive peptides can be used in methods for diagnosing a varicella zoster virus infection.
In accordance with the classification of the International Committee on Taxonomy of Viruses (ICTV), Van Zoute Vin (VZV) is assigned to the Herpesviridae family. In 75% of cases, primary infections take place not later than the age of 15 and usually take an asymptomatic course. By contrast, infection of adults who have not previously had any contact with the virus and in persons who are naturally or therapeutically immunosuppressed can be associated with severe symptoms. Infection of the fetus also leads to severe symptoms since the virus is able to cross the placenta, and maternal antibodies afford no protection at this time. Following primary infection, the virus persists throughout life in sensory ganglia. After reactivation, the VZV spreads over the peripheral nerves in sensory ganglia and then gives rise to herpes zoster.
Seventy open reading frames (ORF), including the open reading frames for the known glycoproteins gpI (ORF 68), gpII (ORF 31), gpIII (ORF 37), gpIV (ORF 67), gpV (ORF 14) and gpVI (ORF 60), can be deduced from the sequence of the VZV genome, which has been completely elucidated and has a length of 124,884 bp (Dumas strain; (A. J. Davison and J. E. Scott (1986), J. Gen. Virol. 67, 1759-1816)). In each case, the amino acid sequence deduced from the nucleotide sequence displays differing degrees of homology with glycoproteins gE, gB, gH, gI, gC and gL of herpes simplex virus (HSV). However, there is nothing to suggest that the sequence homology can also imply a homologous function. The open reading frames of glycoproteins gpI, gpII, gpIII and gpV have been confirmed by means of molecular biology.
Unlike the more thoroughly investigated glycoprotein gB of HSV, few data are available with regard to the homologous protein of VZV, i.e. gpII. The corresponding investigations have been confined to the biosynthesis of gpII and the importance of gpII for virus neutralization (P. M. Keller et al. (1986), Virologie 152, 181-191; M. Masser et al. (1993), J. Gen. Virol. 74, 491-494).
Ellis et al. (E.P. 0210931B1) have confirmed understanding of ORF 31, which is assigned to gpII. In addition, Ellis et al. describe the purification of gpII from VZV-infected MRC 5 cells (human fibroblasts) and its use for preparing virus-neutralizing antibodies from guinea pigs. P. M. Keller et al. (1986), Virologie 152, 181-191 and M. Masser et al. (1993), J. Gen. Virol. 74, 491-494 make use of, inter alia, gpII, which was purified from VZV-infected cells by means of affinity chromatography, for determining the VZV-specific antibody titer of human sera. Following deletion of the carboxyterminal region, A. Bollen (E.P. 0405867 A1; U.S. Pat. No. 371,772) expressed gpII, which lacked its membrane anchor, using the baculovirus expression system in SF9 cells and, by means of constitutive expression in CHO cells, for preparing a VZV vaccine. Furthermore, E. H. Wasmuth and W. J. Miller ((1990), J. Med. Virol. 32, 189-193) used glycoproteins (including gpII), which have been affinity-purified from VZV-infected cells in a glycoprotein or dot ELISA, for demonstrating sensitivity of these test methods and also that antibody titers produced correlated with protection against VZV infection. However, none of the abovementioned publications provides information for preparing adequate quantities of glycoprotein gpII to set up a test on a diagnostically relevant scale. Furthermore, no suitable investigation of the detailed structure of gpII or, in particular, immunoreactive epitopes on the gpII protein have been disclosed.
There are a very wide variety of serological methods for examining the status of VZV immunity. These methods range from radioimmunoassay (RIA), enzyme-linked immunosorbentassay (ELISA), fluorescent antibody membrane antigen assay (FAMA) and immunofluorescence test (IFT) to complement fixation (CF). These methods mainly detect VZV-specific antibodies. Virus material that has been isolated after elaborate culture on human fibroblast cultures and purified for diagnostic use by special methods is frequently employed as the antigen.
Unfortunately, the isolation of VZV antigens from infected fibroblasts is dangerous and carries the danger of infecting laboratory personnel. In addition, the preparation is very costly and time-consuming because, inter alia, the virus is not released from the infected cells and special purification methods are required. In order to use the antigen for an immunochemical test without prior purification, the VZV-infected cells are disrupted by ultrasonication and the antigen is used directly, after dilution, for coating microtitration plates, for example. Use of this method can give rise to false-positive results, and thus incorrect diagnosis. This is because cell-specific antigens (for example, from autoimmune disease), generally also are bound to the solid phase (in this case microtitration plate wells) along with virus-specific antigens. Other test methods based on purified viral glycoproteins, such as glycoprotein ELISA, require purification methods that are markedly more elaborate and involve much greater loss. Thus, it has scarcely been feasible to set up immunochemical diagnostic tests on a relatively large scale. Cross reactivities with HSV-specific antibodies, and false-positive results due to this, are also frequently observed in glycoprotein ELISA, due to marked homology between glycoproteins of the xcex1-herpesviruses.
In one embodiment of the invention, an immunoreactive peptide is provided that is homologous with the AA 450 to 655 region of VZV gpII. In another embodiment, the peptide essentially comprises the region encompassing AA 450 to 655 of gpII of VZV. In another embodiment, the region corresponds to AA 505 to 647 and in another embodiment, the region corresponds to AA 517 to 597. In another embodiment, the region corresponds to AA 535 to 584 and in another embodiment the region corresponds to AA 545 to 582.
Other embodiments of the invention provide nucleic acid that hybridizes under stringent conditions with the nucleic acid depicted in FIG. 2 and which encodes a peptide that is recognized by antibodies which are directed against VZV but not by antibodies which are directed against other herpes viruses. In further embodiments, the invention provides an immunochemical method which uses an immunoreactive peptide as claimed herein for detecting antibodies against VZV in a sample and test kits that contain one or more of the peptides or nucleic acids as described herein. Other embodiments of the invention are readily appreciated, as described further in the specification.